Attaching zanamivir to a polymer markedly enhances its activity against drug-resistant strains of influenza a virus

Effects of the commercial drug zanamivir (Relenza™) covalently attached to poly-l-glutamine on the infectivity of influenza A viruses are examined using the plaque reduction assay and binding affinity to viral neuraminidase (NA). These multivalent drug conjugates exhibit (i) up to a 20,000-fold improvement in anti-influenza potency compared with the zanamivir parent against human and avian viral strains, including both wild-type and drug-resistant mutants, and (ii) superior neuraminidase (NA) inhibition constants, especially for the mutants. These findings provide a basis for exploring polymer-attached inhibitors as more efficacious therapeutics, particularly against drug-resistant influenza strains.National Institutes of Health (U.S.) (Grant Number U01-AI074443)Fundación Ramón Areces. Postdoctoral Fellowshi

Neuraminidase Inhibitor Susceptibility Testing in Human Influenza Viruses: A Laboratory Surveillance Perspective

Neuraminidase inhibitors (NAIs) are vital in managing seasonal and pandemic influenza infections. NAI susceptibilities of virus isolates (n = 5540) collected during the 2008–2009 influenza season were assessed in the chemiluminescent neuraminidase inhibition (NI) assay. Box-and-whisker plot analyses of log-transformed IC50s were performed for each virus type/subtype and NAI to identify outliers which were characterized based on a statistical cutoff of IC50 >3 interquartile ranges (IQR) from the 75th percentile. Among 1533 seasonal H1N1 viruses tested, 1431 (93.3%) were outliers for oseltamivir; they all harbored the H275Y mutation in the neuraminidase (NA) and were reported as oseltamivir-resistant. Only 15 (0.7%) of pandemic 2009 H1N1 viruses tested (n = 2259) were resistant to oseltamivir. All influenza A(H3N2) (n = 834) and B (n = 914) viruses were sensitive to oseltamivir, except for one A(H3N2) and one B virus, with D151V and D197E (D198E in N2 numbering) mutations in the NA, respectively. All viruses tested were sensitive to zanamivir, except for six seasonal A(H1N1) and several A(H3N2) outliers (n = 22) which exhibited cell culture induced mutations at residue D151 of the NA. A subset of viruses (n = 1058) tested for peramivir were sensitive to the drug, with exception of H275Y variants that exhibited reduced susceptibility to this NAI. This study summarizes baseline susceptibility patterns of seasonal and pandemic influenza viruses, and seeks to contribute towards criteria for defining NAI resistance

Inhibition of Neuraminidase Inhibitor-Resistant Influenza Virus by DAS181, a Novel Sialidase Fusion Protein

Antiviral drug resistance for influenza therapies remains a concern due to the high prevalence of H1N1 2009 seasonal influenza isolates which display H274Y associated oseltamivir-resistance. Furthermore, the emergence of novel H1N1 raises the potential that additional reassortments can occur, resulting in drug resistant virus. Thus, additional antiviral approaches are urgently needed. DAS181 (Fludase®), a sialidase fusion protein, has been shown to have inhibitory activity against a large number of seasonal influenza strains and a highly pathogenic avian influenza (HPAI) strain (H5N1). Here, we examine the in vitro activity of DAS181 against a panel of 2009 oseltamivir-resistant seasonal H1N1 clinical isolates. The activity of DAS181 against nine 2009, two 2007, and two 2004 clinical isolates of seasonal IFV H1N1 was examined using plaque number reduction assay on MDCK cells. DAS181 strongly inhibited all tested isolates. EC50 values remained constant against isolates from 2004, 2007, and 2009, suggesting that there was no change in DAS181 sensitivity over time. As expected, all 2007 and 2009 isolates were resistant to oseltamivir, consistent with the identification of the H274Y mutation in the NA gene of all these isolates. Interestingly, several of the 2007 and 2009 isolates also exhibited reduced sensitivity to zanamivir, and accompanying HA mutations near the sialic acid binding site were observed. DAS181 inhibits IFV that is resistant to NAIs. Thus, DAS181 may offer an alternative therapeutic option for seasonal or pandemic IFVs that become resistant to currently available antiviral drugs

Evaluation of the antigenic relatedness and cross-protective immunity of the neuraminidase between human influenza A (H1N1) virus and highly pathogenic avian influenza A (H5N1) virus

AbstractTo determine the genetic and antigenic relatedness as well as the cross-protective immunity of human H1N1 and avian H5N1 influenza virus neuraminidase (NA), we immunized rabbits with either a baculovirus-expressed recombinant NA from A/Beijing/262/95 (BJ/262) H1N1 or A/Hong Kong/483/97 (HK/483) H5N1 virus. Cross-reactive antibody responses were evaluated by multiple serological assays and cross-protection against H5N1 virus challenge was evaluated in mice. In a neuraminidase inhibition (NI) test, the antisera exhibited substantial inhibition of NA activity of the homologous virus, but failed to inhibit the NA activity of heterologous virus. However, these antisera exhibited low levels of cross-reactivity measured by plaque size reduction, replication inhibition, single radial hemolysis, and ELISA assays. Passive immunization with HK/483 NA-specific antisera significantly reduced virus replication and disease, and afforded almost complete protection against lethal homologous virus challenge in mice. However, passive immunization with BJ/262 (H1N1) NA-specific antisera was ineffective at providing cross-protection against lethal H5N1 virus challenge and only slightly reduced weight loss. Substantial amino acid variation among the NA antigenic sites was observed between BJ/262 and HK/483 virus, which was consistent with the lack of cross-reactive NI activity by the antibody and limited cross-protective immunity in mice. These results show a strong correlation between the lack of cross-protective immunity and low structural similarities of NA from a human seasonal H1N1 virus and an avian H5N1 influenza virus

Antiviral susceptibility of clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses isolated from birds and mammals in the United States, 2022

Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) A(H5N1) viruses that are responsible for devastating outbreaks in birds and mammals pose a potential threat to public health. Here, we evaluated their susceptibility to influenza antivirals. Of 1,015 sequences of HPAI A(H5N1) viruses collected in the United States during 2022, eight viruses (∼0.8%) had a molecular marker of drug resistance to an FDA-approved antiviral: three adamantane-resistant (M2-V27A), four oseltamivir-resistant (NA-H275Y), and one baloxavir-resistant (PA-I38T). Additionally, 31 viruses contained mutations that may reduce susceptibility to inhibitors of neuraminidase (NA) (n = 20) or cap-dependent endonuclease (CEN) (n = 11). A panel of 22 representative viruses was tested phenotypically. Overall, clade 2.3.4.4b A(H5N1) viruses lacking recognized resistance mutations were susceptible to FDA-approved antivirals. Oseltamivir was least potent at inhibiting NA activity, while the investigational NA inhibitor AV5080 was most potent, including against NA mutants. A novel NA substitution T438N conferred 12-fold reduced inhibition by zanamivir, and in combination with the known marker N295S, synergistically affected susceptibility to all five NA inhibitors. In cell culture-based assays HINT and IRINA, the PA-I38T virus displayed 75- to 108-fold and 37- to 78-fold reduced susceptibility to CEN inhibitors, baloxavir and the investigational AV5116, respectively. Viruses with PA-I38M or PA-A37T showed 5- to 10-fold reduced susceptibilities. As HPAI A(H5N1) viruses continue to circulate and evolve, close monitoring of drug susceptibility is needed for risk assessment and to inform decisions regarding antiviral stockpiling

Outbreak of Antiviral Drug–Resistant Influenza A in Long-Term Care Facility, Illinois, USA, 2008

An outbreak of oseltamivir-resistant influenza A (H1N1) occurred in a long-term care facility. Eight (47%) of 17 and 1 (6%) of 16 residents in 2 wards had oseltamivir-resistant influenza A virus (H1N1) infections. Initial outbreak response included treatment and prophylaxis with oseltamivir. The outbreak abated, likely because of infection control measures

Influenza in Outpatient ILI Case-Patients in National Hospital-Based Surveillance, Bangladesh, 2007–2008

Recent population-based estimates in a Dhaka low-income community suggest that influenza was prevalent among children. To explore the epidemiology and seasonality of influenza throughout the country and among all age groups, we established nationally representative hospital-based surveillance necessary to guide influenza prevention and control efforts.We conducted influenza-like illness and severe acute respiratory illness sentinel surveillance in 12 hospitals across Bangladesh during May 2007–December 2008. We collected specimens from 3,699 patients, 385 (10%) which were influenza positive by real time RT-PCR. Among the sample-positive patients, 192 (51%) were type A and 188 (49%) were type B. Hemagglutinin subtyping of type A viruses detected 137 (71%) A/H1 and 55 (29%) A/H3, but no A/H5 or other novel influenza strains. The frequency of influenza cases was highest among children aged under 5 years (44%), while the proportions of laboratory confirmed cases was highest among participants aged 11–15 (18%). We applied kriging, a geo-statistical technique, to explore the spatial and temporal spread of influenza and found that, during 2008, influenza was first identified in large port cities and then gradually spread to other parts of the country. We identified a distinct influenza peak during the rainy season (May–September).Our surveillance data confirms that influenza is prevalent throughout Bangladesh, affecting a wide range of ages and causing considerable morbidity and hospital care. A unimodal influenza seasonality may allow Bangladesh to time annual influenza prevention messages and vaccination campaigns to reduce the national influenza burden. To scale-up such national interventions, we need to quantify the national rates of influenza and the economic burden associated with this disease through further studies

Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2015-2016.

Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) assessed antiviral susceptibility of 14,330 influenza A and B viruses collected by WHO-recognized National Influenza Centres (NICs) between May 2015 and May 2016. Neuraminidase (NA) inhibition assay was used to determine 50% inhibitory concentration (IC50) data for NA inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. Furthermore, NA sequences from 13,484 influenza viruses were retrieved from public sequence databases and screened for amino acid substitutions (AAS) associated with reduced inhibition (RI) or highly reduced inhibition (HRI) by NAIs. Of the viruses tested by WHO CCs 93% were from three WHO regions: Western Pacific, the Americas and Europe. Approximately 0.8% (n = 113) exhibited either RI or HRI by at least one of four NAIs. As in previous seasons, the most common NA AAS was H275Y in A(H1N1)pdm09 viruses, which confers HRI by oseltamivir and peramivir. Two A(H1N1)pdm09 viruses carried a rare NA AAS, S247R, shown in this study to confer RI/HRI by the four NAIs. The overall frequency of A(H1N1)pdm09 viruses containing NA AAS associated with RI/HRI was approximately 1.8% (125/6915), which is slightly higher than in the previous 2014-15 season (0.5%). Three B/Victoria-lineage viruses contained a new AAS, NA H134N, which conferred HRI by zanamivir and laninamivir, and borderline HRI by peramivir. A single B/Victoria-lineage virus harboured NA G104E, which was associated with HRI by all four NAIs. The overall frequency of RI/HRI phenotype among type B viruses was approximately 0.6% (43/7677), which is lower than that in the previous season. Overall, the vast majority (>99%) of the viruses tested by WHO CCs were susceptible to all four NAIs, showing normal inhibition (NI). Hence, NAIs remain the recommended antivirals for treatment of influenza virus infections. Nevertheless, our data indicate that it is prudent to continue drug susceptibility monitoring using both NAI assay and sequence analysis